Process for recovering antibiotics U-50,147 and U-51,738

ABSTRACT

Microbiological process for preparing the known antibiotics, antibiotic A-396-1 and L-dihydrophenylalanine, by use of the new microorganism Streptomyces lemensis, NRRL 8170 using controlled fermentation conditions. These antibiotics are active against Gram-positive and Gram-negative bacteria. Accordingly, they can be used in various environments to eradicate or control such bacteria.

This is a division of application Ser. No. 684,014, filed May 7, 1976,now U.S. Pat. No. 4,019,548.

BACKGROUND OF THE INVENTION

Antibiotic A-396-1 is a known antibiotic described in The Journal ofAntibiotics, Vol. 23, No. 6, pages 291-294 (1970) and The Journal ofAntibiotics, Vol. 23, No. 11, pages 569-571 (1970). AntibioticL-dihydrophenylalanine is a known antibiotic described in J. Org. Chem.,33, 1777 (1968).

BRIEF SUMMARY OF THE INVENTION

The novel process of the invention consists of using the newmicroorganism Streptomyces lemensis, NRRL 8170, and employing controlledfermentation conditions, to produce the known antibiotic A-396-1,hereinafter referred to as U-50,147, and L-dihydrophenylalanine,hereinafter referred to as U-51,738.

The antibiotics produced by the subject process have the property ofadversely affecting the growth of Grampositive bacteria, for example,Staphylococcus aureus and Bacillus subtilis, and Gram-negative bacteria,for example, Escherichia coli and Klebsiella pneumoniae. Accordingly,these antibiotics can be used to disinfect washed and stacked foodutensils contaminated with S. aureus; they also can be used asdisinfectants on various dental and medical equipment contaminated withS. aureus. Further, these antibiotics can be used for suppressing thegrowth of sensitive organisms in plate assays and other biologicalmedia.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism used for the production of antibiotics U-50,147 andU-51,738 is streptomyces lemensis, NRRL 8170.

A subculture of this microorganism can be obtained from the permanentcollection of the Northern Regional Research Laboratory, U.S. Departmentof Agriculture, Peoria, Illinois, U.S.A. Its accession number in thisdepository is NRRL 8170. It should be understood that the availabilityof the culture does not constitute a license to practice the subjectinvention in derogation of patent rights granted with the subjectinstrument by governmental action.

The microorganism of this invention was studied and characterized byAlma Dietz of the Upjohn Research Laboratories.

A streptomycete isolated from a soil sample has been studied and foundto differ in macroscopic, microscopic and bichemical properties frommembers of the Genus Streptomyces characterized in Bergey's Manual ofDeterminative Bacteriology [Buchanan, R. E., and N. E. Gibbons. 1974.Bergey's Manual of Determinative Bacteriology, Eight Edition, TheWilliams and Wilkins Co., Baltimore], from cultures characterized inShirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1968.Cooperative description of type cultures of Streptomyces. II. Speciesdescriptions from first study. 18:68-189] [Shirling, E. B., and D.Gottlieb. 1968. Cooperative description of type cultures ofStreptomyces. III. Additional species descriptions from first and secondstudies. Int. J. Syst. Bacteriol. 18:280-399] [Shirling, E. B., and D.Gottlieb. 1969. Cooperative description of type cultures ofStreptomyces. IV. Species descriptions from the second, third and fourthstudies. Int. J. Syst. Bacteriol. 19:391-512] [Shirling, E. B., and D.Gottlieb. 1972. Cooperative description of type strains of Streptomyces.V. Additional descriptions. Int. J. Syst. Bacteriol. 22:285-394], andfrom "type" cultures in the Upjohn Culture Collection.

S. lemensis could be placed in a Gray or Red series of melanin-negativecultures with smooth spores in spiral spore chains. The new culture isreadily distinguished from the cultures in the references cited by itscolor pattern, its utilization of carbon compounds in synthetic media[Pridham, T. G., and D. Gottlieb. 1948. The utilization of carboncompounds by some Actinomycetales as an aid for species determination.J. Bacteriol. 56:107-114] [Shirling, E. B., and D. Gottlieb. 1966.Methods for characterization of Streptomyces species. Int. J. Syst.Bacteriol. 16:313-340], and its production of the antibioticsN-demethylhygromycin B and dihydrophenylalanine.

N-demethylhygromycin B (A-396-1) is produced by Streptoverticilliumeurocidicus, Junichi Shoji, et al. 1970. Isolation of a newwater-soluble basic antibiotic A-396-1, J. Antibiotics 23, 291-294; andTamura, Atsushi, Ryuje Furuta and Hirotada Kotami. 1975. AntibioticA-16316-C, a new water-soluble basic antibiotic. The Journal ofAntibiotics XXVIII: 260-265. The verticillate spore chains of S.eurocidicus make additional comparisons with S. lemensis unnecessary.

Production of dihydrophenylalanine by an actinomycete has not beenreported in the literature.

The distinctive properties cited for the culture characterized requireit to be considered a new species of Streptomyces designatedStreptomyces lemensis sp.n. This is to be considered the type species.Should variants be designated, the type species will become the typesubspecies (Streptomyces lemensis sub.sp. lemensis). The culture isdesignated a new species in accordance with the rules of nomenclatureset forth in the International Code of Nomenclature of Bacteria [Lapage,S. P., P. H. A. Sneath, E. F. Lessel, V. B. D. Sherman, H. P. R.Seeliger and W. A. Clark. 1975. International Code of Nomenclature ofBacteria. Amer. Soc. for Microbiology, Washington, D.C.].

Streptomyces lemensis Dietz sp.n.

Color characteristics.

Aerial growth lavender-gray-tan or grayish reddish brown.Melanin-negative. Appearance on Ektachrome is given in Table 1.Reference color characteristics are given in Table 2. The culture may beplaced in the Red (R) or Gray (GY) color groups of Tresner and Backus[Tresner, H. D., and E. J. Backus. 1963. System of color wheels forstreptomycete taxonomy. Appl. Microbiol. 11:335-338].

Microscopic characteristics.

Smooth spores with convolutions are found in spiral spore chains. Thechains contain more then 10 appressed spores. Microscopic determinationsfollowed the procedure of Pridham et al. [Pridham, T. G., C. W.Hesseltine, and R. G. Benedict. 1958. A guide for the classification ofstreptomycetes according to selected groups. Placement of strains inmorphological sections. Appl. Microbiol. 6:52-79] and Dietz and Mathews[Dietz, A., and J. Mathews. 1971. Classification of Streptomyces sporesurfaces into five groups. Appl. Microbiol. 21:527-533].

Cultural and biochemical characteristics.

Cultural and biochemical characteristics are cited in Table 3.

Carbon utilization.

Carbon utilization was determined following the procedures of Pridhamand Gottlieb, supra, J. Bacteriol. 56:107-114, and Shirling andGottlieb, supra, Int. J. Syst. Bacteriol. 16:313-340. In the former theculture grew well on the basal medium (no carbon compound added) andD-xylose, L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose,maltose, sucrose, lactose, cellobiose, raffinose, dextrin, solublestarch, glycerol, D-mannitol, sodium acetate, sodium citrate, and sodiumsuccinate; moderately on rhamnose, inulin, dulcitol, D-sorbitol,inositol, and sodium tartrate; and poorly on sodium oxalate. There wasno growth on salicin, phenol, cresol, sodium formate, or sodiumsalicylate. In the latter growth was strong on the positive control(basal medium plus D-glucose), D-xylose, D-mannitol, and D-fructose;positive on the negative control (basal medium without carbon compound),L-arabinose, sucrose, rhamnose, and raffinose; and negative on inositoland cellulose.

Temperature.

The culture had good vegetative and aerial growth at 18° to 37° C. Therewas smooth vegetative growth after 24 hours at 45° C. The culture didnot grow at 55° C. The media used for temperature studies wereBennett's, Czapek's sucrose, maltose-tryptone, and Hickey-Tresner agars.

                  TABLE 1                                                         ______________________________________                                        Appearance of Streptomyces lemensis on Ektachrome*                            Agar Medium                                                                              Surface        Reverse                                             ______________________________________                                        Bennett's  Lavender-gray-red                                                                            Gray-red-tan                                        Czapek's sucrose                                                                         Trace lavender-gray                                                                          Trace gray-yellow                                   Maltose-tryptone                                                                         Lavender-gray-red                                                                            Red-tan                                             Peptone-iron                                                                               --           Yellow                                              0.1% Tyrosine                                                                            Trace lavender-gray                                                                          Pale Yellow                                         Casein-starch                                                                            Lavender-gray  Pale lavender-gray                                  ______________________________________                                         *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete            classification. Ann. N.Y. Acad, Sci. 60:152-154.                         

                                      Table 2                                     __________________________________________________________________________    Reference Color Characteristics of Streptomyces lemensis                             De-                                                                           ter-                                                                          mi-   ISCC-NBS Color-Name Charts                                              na-                                                                              Chip                                                                             illustrated with Centroid Colors*                                Agar Medium                                                                          tion                                                                             No.                                                                              Color                                                            __________________________________________________________________________    Bennett's                                                                            S  45 l.gy. r Br                                                                           Light Grayish Reddish Brown                                      R  75 deep y Br                                                                            Deep Yellowish Brown                                                73 d. y. Br                                                                             Dark Yellowish Brown                                             P  79 l.gy. yBr                                                                            Light Grayish Yellowish Brown                             Czapek's                                                                             S  73 p. OY  Pale Orange Yellow                                        sucrose                                                                              R  73 p. OY  Pale Orange Yellow                                               P  -- --     --                                                        Maltose-                                                                             S  45 l.gy. r Br                                                                           Light Grayish Reddish Brown                               tryptone                                                                             R  76 ly.y Br                                                                              Light Yellowish Brown                                            P  79 l. gy. y Br                                                                          Light Grayish Yellowish Brown                             Hickey-                                                                              S  45 l.gy. r Br                                                                           Light Grayish Reddish Brown                               Tresner                                                                              R  68 s. OY  Strong Orange Yello                                              P  79 l.gy. y Br                                                                           Light Grayish Yellowish Brown                             Yeast extract-                                                                       S  45 l.gy. r Br                                                                           Light Grayish Reddish Brown                               malt extract                                                                         R  17 m. OY  Moderate Orange Yellow                                    (ISP-2)                                                                              P  79 l.gy. y Br                                                                           Light Grayish Yellowish Brown                             Oatmeal                                                                              S  45 l.gy. r BR                                                                           Light Grayish Reddish Brown                               (ISP-3)                                                                              R  67 brill. OY                                                                            Brilliant Orange-Yellow                                          P  -- --     --                                                        Inorganic-                                                                           S  45 l.gy. r Br                                                                           Light Grayish Reddish Brown                               salts-starch                                                                         R  74 s. y Br                                                                              Strong Yellowish Brown                                    (ISP-4)                                                                              P  72 d. OY  Dark Orange Yellow                                        Glycerol-                                                                            S  60 l.gy. Br                                                                             Light Grayish Reddish Brown                               asparagine                                                                           R  71 ml OY  Moderate Orange Yellow                                    (ISP-5)   67 brill. OY                                                                            Brilliant Orange Yellow                                          P  79 l.gy. y Br                                                                           Light Grayish Yellowish Brown                             __________________________________________________________________________     S = Surface                                                                   R = Reverse                                                                   P = Pigment                                                                   *Kelly, K.L., and D.B. Judd. 1955. The ISCC-NBS method of designating         colors and a dictionary of color names. U.S. Dept. of Comm. Circ. 553,        Washington, D.C.                                                         

                                      Table 3                                     __________________________________________________________________________    Cultural and Biochemical Characteristics of Streptomyces lemensis             Medium   Surface     Reverse   Other Characteristics                          __________________________________________________________________________     Agar                                                                         Peptone-iron                                                                           --          Pale colorless                                                                          No pigment                                                          or cream  Melanin negative                               Calcium malate                                                                         --          Colorless No pigment                                                                    Malate not solubilized                         Glucose  Lavender-gray-cream                                                                       Orange-tan                                                                              Pale orange-tan pigment                        asparagine                                                                    Skim milk                                                                              Trace gray-white                                                                          Pale orange-tan                                                                         Pale orange-tan pigment                                                       Casein solubilized under                                                      growth to around growth                        Tyrosine Pale gray-pink                                                                            Pale cream                                                                              Pale cream pigment                                                            Tyrosine not solubilized                       Xanthine Trace gray-pink                                                                           Very pale cream                                                                         Pale cream pigment                                                            Xanthine solubilized                           Nutrient starch                                                                        Pale gray-pink                                                                            Pale cream                                                                              No pigment                                                                    Starch partially solubilized                   Yeast extract-                                                                         Good lavender-gray                                                                        Red-tan with                                                                            Pink-tan pigment                               malt extract         pale tan edge                                            Peptone-yeast                                                                          --          Pale yellow-tan                                                                         Pale yellow-tan pigment                        extract-iron                                                                  (ISP-6)                                                                       Tryosine (ISP-7)                                                                       Good lavender-gray                                                                        Pale red-tan                                                                            Pale red-tan pigment                           Gelatin                                                                       Plain    --          --        Liquefaction - 1/4                             Nutrient --          --        Liquefaction - 1/4 to                                                         complete                                       Broth                                                                         synthetic nitrate                                                                      Colorless flaky                                                                           --        No pigment                                              surface pellicle      Trace growth throughout                                                       Flocculent bottom growth                                                      Nitrate reduced to nitrite                     Nutrient nitrate                                                                       Colorless surface                                                                         --        Flocculent bottom growth                                pellicle              Nitrites not found                             Litmus milk                                                                            Gray aerial growth                                                                        Blue-gray Peptonization - 2/3                                     or surface ring       Coagulation - 1/3                                                             Decolorization - 1/3                                                          pH 6.73-7.55                                   __________________________________________________________________________

The compounds of the invention process are produced when the elaboratingorganism is grown in an aqueous nutrient medium under submerged aerobicconditions. It is to be understood, also, that for the preparation oflimited amounts surface cultures and bottles can be employed. Theorganism is grown in a nutrient medium containing a carbon source, forexample, an assimilable carbohydrate, and a nitrogen source, forexample, an assimilable nitrogen compound or proteinaceous material.Preferred carbon sources include glucose, brown sugar, sucrose,glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like.Preferred nitrogen sources include cornsteep liquor, yeast, autolyzedbrewer's yeast with milk solids, soybean meal, cottonseed meal,cornmeal, milk solids, pancreatic digest of casein, fish meal,distillers' solids, animal peptone liquors, meat and bone scraps, andthe like. Combinations of these carbon and nitrogen sources can be usedadvantageously. Trace metals, for example, zinc, magnesium, manganese,cobalt, iron, and the like, need not be added to the fermentation mediasince tap water and unpurified ingredients are used as components of themedium prior to sterilization of the medium.

Production of the compounds by the invention process can be effected atany temperature conducive to satisfactory growth of the microorganism,for example, between about 18° and 40° C., and preferably between about20° and 28° C. Ordinarily, optimum production of the compound isobtained in about 3 to 15 days. The medium normally remains acidicduring the fermentation. The final pH is dependent, in part, on thebuffers present, if any, andin part on the initial pH of the culturemedium.

When growth is carried out in large vessels and tanks, it is preferablyto use the vegetative form, rather than the spore form, ofthemicroorganism for inoculation to avoid a pronounced lag in theproduction of the compounds and the attendant inefficient utilization ofthe equipment. Accordingly, it is desirable to produce a vegetativeinoculum in a nutrient broth culture by inoculating this broth culturewith an aliquot from a soil, liquid N₂ agar plug, or a slant culture.When a young, active vegetative inoculum has thus been secured, it istransferred aseptically to large vessels or tanks. The medium in whichthe vegetative inoculum is produced can be the same as, or differentfrom, that utilized for the production of the compounds, so long as agood growth of th emicroorganism is obtained.

A variety of procedures can be employed in the isolation andpurification of the compounds produced by the subject invention fromfermentation beers, for example, solvent extraction, partitionchromatography, silica gel chromatography, liquid-liquid distribution ina Craig apparatus, adsorption on resins, and crystallization fromsolvents.

In a preferred recovery process the compounds produced by the subjectprocess invention are recovered from the culture medium by separation ofthe mycelia and undissolved solids by conventional means, such as byfiltration or centrifugation.

The antibiotics are then recovered from the filtered or centrifugedbroth by absorption on a resin column. Non-ionic (preferred) as well ascationic exchange resins can be used. Both the carboxylic acid andsulfonic acid types can be used. Suitable non-ionic resins includeresins comprising a non-ionic macro porous copolymer of styrenecrosslinked with divinylbenzene. Non-ionic resins of this type aremarketed under the trade names Amberlite XAD-2 and XAD-4, disclosed inU.S. Pat. No. 3,515,717. Suitable carboxylic acid resins include thepolyacrylic acid resins obtained by the copolymerization of acrylic acidand divinylbenzene by the procedure given on page 87 of Kunin, IonExchange Resins, 2nd Ed. (1958), John Wiley and Sons, Inc. Carboxylicacid cation exchange resins of this type are marketed under the tradenames Amberlite IRC-50 and Zeokarb 226. Suitable sulfonic acid resinsinclude sulfonated polystyrene resins crosslinked with divinylbenzeneobtained by the procedure given on page 84 of Kunin, supra. Sulfonatedcation exchange resins of this type are marketed under the trade namesDowex-50, Amberlite IR-120, Nalcite HCR, Chempro C-20, Permutit Q, andZeokarb 225.

In the preferred process, using the non-ionic resin, the filtrate fromthe filtered fermentation beer is passed over the resin. The spent iscollected. The column is then washed with water and the aqueous wash iscollected. This spent and aqueous wash contain Antibiotic U-50,147. Thisantibiotic is recovered from these materials by chromatography over acarboxylic acid cation exchange resin or a sulfonic acid type resin asdescribed above.

The material is passed over the cation exchange resin. The column iseluted with a basic solution, for example 0.25 N aqueous ammoniumhydroxide (preferred), and other bases like 0.25 N aqueous sodiumhydroxide, potassium hydroxide, and pyridinium acetate buffers preparedby mixing pyridine and acetic acid. Antibacterially-active fractions arecollected. The active fractions are purified by use of a strongly basicanion exchange resin to remove impurities. Suitable anion exchangeresins for this purpose are obtained by chlormethylating by theprocedure given on pages 88 and 97 of Kunin,, supra, polystyrenecrosslinked, if desired, with divinylbenzene prepared by the proceduregiven on page 84 of Kunin, supra, and quaternizing with trimethylamine,or dimethylthanolamine by the procedure given on page 97 of Kunin,supra. Anion exchange resins of this type are marketed under the tradenames Dowex-1, Dowex-2, Dowex-3, Dowex-2K, Amberlite IRA-400, AmberliteJR-45, Duolite A-102, and Ionac A-300. Dowex-1(OH) is the preferredresin. Antibacterially-active fractions from the anion exchange resinare concentrated to dryness to give an essentially pure preparation ofAntibiotic U-50,147.

Antibiotic U-51,738 is recovered from the fermentation beers of thesubject invention process by first filtering the beer and then passingthe filtrate over a non-ionic resin as described above. AntibioticU-50,147 is not adsorbed onto the non-ionic resin, but, rather, passesthrough or is washed off the resin by water. On the other hand,antibiotic U-51,738 is adsorbed on the non-ionic resin and is removed byelution with a lower-alkanol-water solution. Preferably, methanol-water(90:10) is used to elute antibiotic U-51,738 from the non-ionic resin.Fractions containing antibiotic U-51,738, as determined by antibacterialtesting hereinafter described, are combined and concentrated in vacuo.Insoluble crystalline material is separated during the concentration togive an essentially pure crystalline preparation of antibiotic U-51,738.This material is then recrystallized from a lower alcohol (ethanol ispreferred) and water, to give an essentially pure crystallinepreparation of antibiotic U-51,738.

Hereinafter are described non-limiting examples of the process of thesubject invention. All percentages are by weight and all solvent mixtureproportions are by volume unless otherwise noted.

EXAMPLE 1 Part A. Fermentation

A soil stock of Streptomyces lemensis, NRRL 8170, is used to inoculate aseries of 500-ml Erlenmeyer flasks, each containing 100 ml of sterileseed medium consisting of the following ingredients:

    ______________________________________                                        Glucose monohydrate  10 g/liter                                               Bacto peptone (Difco)                                                                              10 g/liter                                               Bacto Yeast Extract (Difco)                                                                        2.5 g/liter                                              Deionized Water      Balance                                                  ______________________________________                                    

The seed inoculum is grown for 96 hours at 28° C. on a Gump rotaryshaker operating at 250 rpm and having a 21/2 inch stroke.

Seed inoculum (5%) prepared as described above, is used to inoculate aseries of 500 ml Erlenmeyer fermentation flasks containing 100 ml ofsterile fermentation medium consisting of the following ingredients:

    ______________________________________                                        Cerelose             14 g/liter                                               Starch (Buffalo)*    12.5 g/liter                                             Cottonseed meal**    40 g/liter                                               Tap water q.s.       1 liter                                                  ______________________________________                                         *CPC International, Englewood Cliffs, N.J.                                    **Southern Cotton Oil Division, Hunt Foods and Industries, Newport,           Arkansas.                                                                

The pH of the fermentation medium is adjusted to 7.2 with an aqueoussolution of sodium hydroxide before sterilization. The inoculatedfermentation flasks are incubated at 28° C. on a rotary shaker operatingat 250 rpm. The fermentation is monitored by assaying samples with themicroorganisms Bacillus subtilis and Penicillium oxalicium to give thetotal assay for antibiotics U-50,147 and U-51,738. A representative5-day fermentation has the following titers of antibiotics in thefermentation broth:

    ______________________________________                                                 Assay in BU/ml                                                       Day        B. subtilis   P. oxalicum                                          ______________________________________                                        2          2.0           1.6                                                  3          7.0           2.5                                                  4          10.0          4.0                                                  5          10.0          3.2                                                  ______________________________________                                    

The assay is a disc-plate biounit assay on standard culture medium. TheB. subtilis plates are prepared with Streptomycin Assay Agar with YeastExtract (Antibiotic Medium #5, BBL, Cockeysville, Md.), and the P.oxalcium plates are prepared with Sabouraud Dextrose Agar (DifcoLaboratories, Detroit, Michigan). The B. subtilis assay plates areincubated at 32° C. for 18 hours and the P. oxalicum assay plates areincubated at 28° C. for 18 hours.

A biounit (BU) is defined as the concentration of the antibiotic whichgives a 20 mm zone of inhibition under the above assay condition. Thus,if for example a fermentation beer, or other solution containing theantibiotic, needs to be diluted 1/100 to give a 20 mm zone ofinhibition, the potency of such beer or solution is 100 BU per ml.

Part B. Recovery

(1) Filtration

Fermentation broth, as described above, ca. 10 liters, is filtered withthe aid of diatomaceous earth. The filtrate is then subjected tochromatography over Amberlite XAD-4 as disclosed below.

(2) Chromatography Over Amberlite XAD-4

The column is prepared from 750 ml of Amberlite XAD-4 resin packed inwater. Filtrate, obtained as described above, ca. 7.5 liters, is passedover the column at a rate of 30 ml/min. The spent filtrate is collectedas one fraction (SPENT). The column is then washed with 2 liters ofwater at a rate of 30 ml/min. The aqueous wash is also collected as onefraction (AQUEOUS). The column is then eluted with 90% aqueous methanol.Fractions of 20 ml are collected. Results follow:

    ______________________________________                                                    Zone Size (mm)                                                                B. subtilis                                                                          P. vulgaris                                                                             P. oxalicum                                      ______________________________________                                        Starting Material                                                                           31       17        20                                           (Filtrate)                                                                    Spent         27       0         traces                                       Aqueous       25       0         0                                            90% Methanolic Eluates                                                        Fraction No.                                                                   5            22       0         0                                            10            20       0         0                                            15            20.5     0         0                                            20            22       0         0                                            25            24       0         0                                            30            35       26        42                                           35            33       25        41                                           40            32       23        40                                           45            31       21        34                                           50            29       17        21                                           55            27.5     traces    16                                           60            26       0         traces                                       65            25       0         traces                                       70            24       0         0                                            75            23.5     0         0                                            80            24       0         0                                            85            23       0         0                                            90            23       0         0                                            95            22.5     0         0                                            100           22       0         0                                            ______________________________________                                    

Thin layer chromatography (tlc) using Eastman Cellulose Chromagram(13255) as support and a solvent consisting of 96% H₂ O and 4% butanolis used to analyze or monitor the recovery operation. The presence ofthe desired antibiotics are shown by bioautography using themicroorganisms P. oxalicum, K. pneumoniae, and S. lutea. This tlc showsthat the spent filtrate and the aqueous wash contains antibioticU-50,147. These two fractions are combined and labeled ADA-71A.Methanolic fractions 30-60 from the Amberlite XAD-4 column are shown bytlc to contain antibiotic U-51,738.

Preparations similar to ADA-71A are prepared from additionalfermentation broth to give ca. 36 liters of material containingantibiotic U-50,147. The isolation of U-50,147 from this material isaccomplished by chromatography over Amberlite IRC 50 (H⁺), describedbelow.

(3) Chromatography Over Amberlite IRC-50 (H⁺)

The column is prepared from 1.5 liter of Amberlite IRC-50 in thehydrogen form. The starting material, 36 liters of the combinedfiltrates-wash, is adjusted to pH 3.0 with aqueous sulfuric acid.Insoluble material is separated by filtration. The clear filtrate isadjusted to pH 8.5 with aqueous sodium hydroxide and is passed over thecolumn. The spent is collected as one fraction (SPENT). The column iswashed with 12 liters of water. The aqueous wash is collected as onefraction (AQUEOUS). The column is washed with 6 liters of 0.25 N aqueousammonium hydroxide collected as one fraction (0.25 N AMMONIA). Thecolumn is eluted with 1 N aqueous ammonium hydroxide. Fractions of 20 mlare collected. Results follow:

    __________________________________________________________________________              Zone Size (mm)                                                                B. subtilis                                                                         K. pneumoniae                                                                          P. vulgaris                                                                          P. oxalicum                                   __________________________________________________________________________    Spent     0     0        --     --                                            Aqueous   0     0        --     --                                            0.25 N Ammonia                                                                          0     0        --     --                                            1 N Ammonia                                                                   Fraction No.                                                                  10        0     0        --     --                                            20        0     0        --     --                                            .         .     .        .      .                                             .         .     .        .      .                                             230       0     0        --     --                                            240       21    traces   --     --                                            250       31    24       --     --                                            260       32    26       --     --                                            270       36      28.5   --     --                                            280       37    31       --     --                                            290       39    33       --     --                                            300       40    33       --     --                                            310         43.5                                                                              39       26     32                                            320       43      37.5     25.5 31                                            330       42    38       26     31                                            340         42.5                                                                                36.5   25     30                                            350       37    23       20     23                                            360       19      17.5   0      0                                             370       traces                                                                              0        0      0                                             390       traces                                                                              0        0      0                                             400       0     0        0      0                                             .         .     .        .      .                                             570       0     0        0      0                                             __________________________________________________________________________

Fractions 250-279 are combined and concentrated to dryness in vacuo togive Preparation ADA-116.1, 890 mg.

Fractions 280-350 are treated similarly to give Preparation ADA-116.2,6.28 g.

Preparations ADA-116.1 and ADA-116.2 contain antibiotic U-50,147 only(by tlc). Testing of preparation ADA-116.1 and ADA-116.2 shows thefollowing:

    ______________________________________                                                 Zone Size (mm)                                                                ADA-116.1*  ADA-116.2*                                               ______________________________________                                        P. oxalicum                                                                              30            32                                                   P. vulgaris                                                                              24            26                                                   K. pneumoniae                                                                            36            39                                                   B. subtilis                                                                              39            41                                                   ______________________________________                                         *Solutions of 10 mg/ml in water are tested.                              

Preparations ADA-116.1 and ADA-116.2 are combined and purified byDowex-1 (OH⁻) chromatography as described below.

(4) Chromatography Over Dowex-1 (OH⁻)

The column is prepared from 400 ml of Dowex-1 (Cl⁻) (Dow Chemical Co.,Midland, Michigan).

Six liters of 2 N aqueous sodium hydroxide is passed over the colunn ata rate of 10 ml/min. The column is then washed with water until the pHof the effluent is ca. 8.0.

Preparations ADA-116.1 and ADA-116.2 are combined (ca. 7.1 g) dissolvedin 25 ml of water and passed over the column at a rate of 4 ml/min. Thecolumn is eluted with water. Fractions of 20 ml are collected. Testingshows the following:

    ______________________________________                                                 Zone Size (mm)                                                       Fraction No.                                                                             B. subtilis   K. pheumoniae                                        ______________________________________                                        3          0             0                                                    6          0             0                                                    9          27            20                                                   12         25            18                                                   15         18.5          traces                                               18         16            0                                                    21         traces        0                                                    24         17            0                                                    27         18            traces                                               30         21            traces                                               33         22            15                                                   36         22            15                                                   39         23            16                                                   42         24            16                                                   45         25            17                                                   48         24            17                                                   51         25            18                                                   54         26            19                                                   57         27            20                                                   60         30            23                                                   62         30            22                                                   66         30            22                                                   69         30            22                                                   --         --            --                                                   60         24            20.5                                                 65         25.5          22                                                   70         25.5          21.5                                                 75         26            22.5                                                 80         27            24                                                   85         28            24.5                                                 90         28.5          25.5                                                 95         30            27                                                   100        31            28.5                                                 110        32            27.5                                                 120        32            29                                                   130        33            30                                                   140        33.5          31                                                   150        34            31.5                                                 160        34.5          31                                                   170        35            32                                                   180        35            32                                                   190        35            32                                                   200        35            32                                                   210        35            30                                                   220        34            30                                                   230        33            30                                                   240        32            29                                                   250        31.5          29.5                                                 260        31            29.5                                                 270        31            29                                                   280        30            28.5                                                 290        30            27.5                                                 300        29            27.5                                                 310        28.5          26                                                   320        28            26                                                   330        27            25                                                   340        26            24                                                   350        26.5          23                                                   360        26            22                                                   370        26            22                                                   380        26            22                                                   390        25.5          22                                                   400        26            22.5                                                 410        24            21                                                   420        24            21                                                   430        23            20.5                                                 440        23.5          20                                                   450        23            20                                                   460        23            20                                                   470        23            19                                                   480        23            19                                                   490        23            20                                                   500        22.5          20                                                   510        22.5          20                                                   520        22            19                                                   530        22            19                                                   540        21.5          18.5                                                 550        21            18                                                   ______________________________________                                    

The following pools are made. Each pool is concentrated to dryness togive the following preparations.

    ______________________________________                                        Pool I   Fractions 8-18                                                                              ADA-123.1,      360 mg                                 Pool II  Fractions 30-50                                                                             ADA-123.2,      570 mg                                 Pool III Fractions 51-90                                                                             ADA-123.3,      333 mg                                 Pool IV  Fractions 91-200                                                                            ADA-123.4,      See                                    Pool V   Fractions 201-300                                                                           ADA-123.5,      Below                                  Pool VI  Fractions 301-500                                                                           ADA-123.6,      330 mg                                 ______________________________________                                    

Preparations ADA-123.4 and ADA-123.5 are combined and kept asADA-123.4A, 1.64 G.

Preparation ADA-123.4A contains essentially pure (tlc and paperchromatography) antibiotic U-50,147.

Antibiotic U-50,147 has the following characteristics:

a. Infrared

The IR bands in Nujol and KBr are presented in Tables A (Nujol) and B(KBr) as follows:

                  Table A                                                         ______________________________________                                        Band Tabulation of the Infrared Spectrum of                                   Antibiotic U-50,147 (Nujol Mull)                                              Band Frequency                                                                (Wave Numbers)  Intensity                                                     ______________________________________                                        3350            S                                                             3290            S                                                             2960            N, S                                                          2920            N, S                                                          2850            N, S                                                          2730            M                                                             1640            N, sh                                                         1588            S                                                             1462            N, S                                                          1378            N, S                                                          1368            S, sh                                                         1342            M                                                             1245            M                                                             1152            S                                                             1078            S                                                             1040            S                                                             1008            S                                                              940            M                                                              892            M                                                              860            M                                                              808            M                                                              790            M                                                              722            N, M                                                           670            M                                                             ______________________________________                                         Key:                                                                          S=Strong,                                                                     M=Medium,                                                                     W=Weak,                                                                       sh=Shoulder,                                                                  N=Nujol                                                                  

                  Table B                                                         ______________________________________                                        Band Tabulation of the Infrared Spectrum of                                   Antibiotic U-50,147 (KBr Pellet)                                              Band Frequency                                                                (Wave Numbers)  Intensity                                                     ______________________________________                                        3410            S                                                             3370            S                                                             3290            S, sh                                                         2920            M                                                             2880            M                                                             2700            M, sh                                                         1630            M, sh                                                         1590            M                                                             1458            M                                                             1385            M                                                             1339            M                                                             1242            M                                                             1152            S                                                             1075            S                                                             1038            S                                                             1005            S                                                              938            M                                                              890            M                                                              805            M                                                              790            M                                                              730            M                                                              700            M                                                              668            M                                                              Key:                                                                          S=Strong,                                                                     M=Medium,                                                                     W=Weak,                                                                       sh=Shoulder                                                              

b. UV Absorption Spectrum

Antibiotic U-50,147 does not show any UV maxima between 220-400 mn.

c. Titration Data

Potentiometric titration in water using aqueous hydrochloric acid showsan equivalent weight of 192.

d. Elemental Analyses

Calculated for C₁₉ H₃₅ H₃ O₁₃ : C, 44.44; H, 6.82; N, 8.18; O, 40.55.Found: C, 41.78; H, 6.92; N, 8.64.

e. Molecular Weight

The molecular weight determined by field desorption mass spectrometry isfound to be 513. Field desorption mass spectra are obtained on the CH5DFmass spectrometer.

f. Optical Rotation

[α]_(D) ²⁵ + 13° (C, 0.95, water)

g. Solubilities

Antibiotic U-50,147 (both the free base and the salts) is soluble inwater and lower alcohols (methanol, ethanol). The antibiotic isinsoluble in higher alcohols, acetone, ethyl acetate, chlorinatedhydrocarbon and saturated hydrocarbon solvents.

EXAMPLE 2 Isolation of Antibiotic U-51,738

The methanolic fractions obtained in Example 1, Part B (2) from theAmberlite XAD-4 resin are combined and the solution is concentrated invacuo. Insoluble crystalline material of antibiotic U-51,738 isseparated during the concentration; yield 1.8 grams. 700 mg of thismaterial is recrystallized from 60 ml of ethanol and 10 ml of water togive an essentially pure crystalline preparation of antibiotic U-51,738.The crystalline material is in the form of colorless needles. AntibioticU-51,738 is a known antibiotic, L-dihydrophenylalanine, having thefollowing structural formula: ##STR1##

This compound has the following reported characteristics: (See J. Org.Chem. 33, 1779 [1968]).

Anal. Calcd. for C₉ H₁₃ NO₂ : C, 64.7; H, 7.84; N, 8.38. Found: C,65.0;H, 7.86; N, 8.53.

IR bands: KBr.sup.ν max 3030-2850, 2620, 1590-1560, 1480, 1390, 1220,1135, 965, 895, 855 cm⁻¹.

We claim:
 1. A process for recovering antibiotic U-50,147 from afermentation mixture f antibiotics U-50,147 and U-51,738 whichcomprises:(a) filtering a fermentation beer containing antibioticU-50,147 and antibiotic U-51,738 to obtain a clear beer; (2) passingsaid clear beer over a non-ionic resin and collecting a spent filtrate;(3) washing said resin with water and collecting an aqueous wash; (4)combining said spent and aqueous wash and chromatographing them over acationic exchange resin and eluting said resin with a basic solution toobtain fractions containing antibiotic U-50,147; (5) passing saidfractions containing antibiotic U-50,147 over a strongly basic anionexchange resin and eluting said resin with water and collectingfractions; and (6) concentrating said fractions containing antibioticU-50,147 to give an essentially pure preparation of antibiotic U-50,147.2. A process, according to claim 1, wherein said non-ionic resin isAmberlite XAD-4.
 3. A process, according to claim 1, wherein said cationexchange resin is Amberlite IRC-50 (H⁺).
 4. A process, according toclaim 1, wherein said cation exchange resin is eluted wit 0.25 N aqueousammonium hydroxide.
 5. A process, according to claim 1, wherein saidanion exchange resin is Dowex-1 l (OH⁻).
 6. A process for recoveringantibiotic U-51,738 from a fermentation mixture of antibiotics U-51,738and U-50,147 which comprises:(1) filtering a fermentation beercontaining antibiotic U-51,738 and antibiotic U-50,147 to obtain a clearbeer; (2) passing said clear beer over a non-ionic resin and elutingsaid resin with a lower-alkanol-water solution to obtain fractionscontaining antibiotic U-51,738; and (3) concentrating said fractions togive an essentially pure crystalline preparation of antibiotic U-51,738.7. A process, according to claim 6, wherein said non-ionic resin isAmberlite XAD-4.
 8. A process, according to claim 6, wherein saidlower-alkanol-water solution is methanol-water (90:10).